Immunoassays for detection of viral subtypes and other antigens

ABSTRACT

The invention provides methods for detection and identification of viruses, viral subtypes and other antigens. Polyclonal antibodies specific for a viral subtype are separated and then bound to a solid support. The antibodies are then contacted with a test sample suspected of containing the viral subtype. Virus particles binding to the separated antibodies are then detected and quantified. In other embodiments of the invention, a test sample suspected of containing an antigen is bound to a solid support, and then contacted with polyclonal antibodies specific for the antigen, the antibodies having a detectable label bound thereon. The detectable label is then detected. The invention also provides kits for performing the methods of the invention.

large number of different monoclonals needed to test the virusadequately. Monoclonal antibodies are very time-consuming to produce andthe specificity and cross-reactivities cannot be controlled except bylengthy selection procedures. Also, mutations can not be picked upunless the panel of monoclonal antibodies being used happens to have aspecificity for the critical antigenic determinant.

Despite the efforts which have been made to develop accurate tests fordetecting viruses, tests are required which can be performed accuratelyand with minimal need for specialized equipment. Tests which can beperformed in the field are also needed. Poultry growers are oftenlocated far from research centers and agriculture department stations.Tests which can be performed in the field would allow poultry growers toavoid delay in treating birds and taking precautions for preventing thespread of the virus. Whether in the field or the laboratory, there isalso a great need for tests which can accurately and rapidly distinguishbetween pathological and non-pathological subtypes of avian influenzavirus.

The problems in providing fast accurate tests to detect avian influenzavirus are also encountered in immunoassays for other substances,particularly if the substance contains multiple antigenic determinantsor epitopes, such as would be present in living organisms and largermolecules. Although monoclonal antibodies are valuable fordistinguishing between closely related organisms, immunoassays usingmonoclonal antibodies may give inaccurate results if the test samplecontains an organism not having the epitope corresponding to themonoclonal antibody employed in the assay. This situation could easilyarise if there has been a mutation in the organism deleting or modifyingthe antigenic determinant.

It is an object of this invention to provide novel methods fordetection, identification and quantitation of antigens and hence thesource or organism containing the antigen, such as viral subtypes andstrains. It is also an object of the invention to provide novel methodsfor detecting, identifying and quantitating avian influenza virussubtypes and strains. It is a further object of the invention to providekits for use in detecting, identifying and quantitating antigens andhence the organism containing the antigen, such as viruses, viralsubtypes and strains.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1c show the dot blot assay for avian influenza virus strainH5N2/Mal/NY/82, FIGS. 1b and 1d show the dot blot assay for avianinfluenza strain H6N8/Ty/Mn/83 and FIG. 1e shows the dot blot system forH2N7/Ty/Mn/86.

FIGS. 2a and 2b show quantatitive representation of two viral proteinsafter computer-analyzed digitization.

FIG. 3A show a transblot of hyperimmune sera of two rabbits injectedwith avian influenza H5N2/Mal/NY/82 where the virus is adsorbed ontonitrocellulose and probed with biotinylated antibodies and FIG. 3B showstransblot of three different subtypes of avian influenza virus analyzedwith a single virus to determine epitope differences among the subtypes.

FIGS. 4a and 4b show line and bar graphs, respectively, of the bands oflane 4 in FIG. 3a that have been quantitated by densitometry andcomputer-digitized. FIGS. 4c and 4d show line and bar graphs,respectively, of the bands of lane 5 in FIG. 3a that have beenquantitated by densitometry and computer-digitized.

SUMMARY OF THE INVENTION

The invention provides methods of detecting an antigen in a test samplesuspected of containing said antigen, and hence the source or organismcontaining the antigen. Polyclonal antibodies specific for the antigenare separated and then applied to a solid support. The separatedantibodies are then contacted with a test sample suspected of containingthe antigen under conditions selected to allow binding of the antigen tothe antibodies. Antigen bound to the antibodies on the solid support isthen contacted with antibodies specific for the antigen under conditionsselected to allow binding of the antibodies to the bound antigen. Theantibodies to the antigen used in this step have a detectable labelbound thereon. The detectable label is then detected. A preferredantigen is viral protein; a preferred virus is avian influenza virus.

The methods may further comprise the step of removing antibodiescross-reactive with antigens other than the antigen being tested fromthe polyclonal antibodies before the first separating step. The methodsmay also comprise the step of quantitating the amount of detectablelabel thereby quantitating the amount of the antigen present in the testsample and thus the amount of the organism containing the antigen in thetest sample. A preferred method of quantitation is by densitometricmeasurement. Methods of separating antibodies known in the art aresuitable for use in the invention. A preferred method is isoelectricfocusing because this method accurately separates antibodies inpolyclonal antisera into bands containing only one type of antibody,i.e. each band acts like a separate "monoclonal" antibody.

The methods of the invention are suitable for use with detectable labelsand detection systems and methods known in the art. A preferreddetectable label is biotin and a preferred method for detecting thedetectable label comprises the steps contacting the biotin withstreptavidin-alkaline phosphatase under conditions selected to allow thestreptavidin-alkaline phosphatase to bind to the biotin; contacting thestreptavidin-alkaline phosphatase with a chromogenic substrate; anddetecting a reaction product of the chromogenic substrate.

Part of the mammalian immunologic response to an antigen is to produceantibodies specific for the antigen. An antigen is material that elicitsantibody production. Proteins, polysaccharides and nucleic acids areusually effective antigens. The specificity of an antibody is directedagainst a particular site on an antigen called the antigenicdeterminant. An antigen may contain multiple antigenic determinants. Inproteins, an antigenic determinant may comprise only six or seven aminoacids of the protein. As used herein, the term antigen includesproteins, polysaccharides, nucleic acids and other cellular componentscapable of provoking an immune response, as well as microorganisms, suchas viruses, which are also capable of provoking an immune response. Theterm antigen also includes substances not capable of provoking anantigenic response, i.e. haptens, but which are capable of provoking anantigenic response when conjugated with a carrier molecule.

A typical virus, for example, has multiple antigenic determinants withinthe proteins which make up its outer envelope (as well as internally)that provoke a multitude of different antibodies in the host mammal. Inaddition, different antibodies are produced to each antigenicdeterminant in a single macromolecular antigen, depending upon whichpart of the antigen provoked the antibody response. The sum of thisresponse to the virus theoretically represents antibodies made to allthe antigenic determinants present on the virus, including those sharedby other viruses and those belonging only to that particular viral typeor subtype. When these polyclonal antibodies are separated by techniquessuch as isoelectric focusing or other techniques to form bands ofantibodies each having its own identifiable electric charge, andvisualized; the antibodies form a "fingerprint" of the virus which canbe used to distinguish it from other viruses or viral subtypes andstrains. The use of a spectrum of antibodies to the virus overcomesproblems associated with monoclonal antibodies such as the need for anumber of monoclonal antibodies to adequately type the virus, loss ofspecificity through mutation of the virus, or lack of specificity fordifferent isolates of the same viral subtype. By using a wide variety ofantibodies to one virus, a change in one or a small number of antibodiesis less noticeable overall. This ensures that the results of detectionmethods using the viral "fingerprint" will remain reliable and accurateeven if some changes in the virus occur. Additionally, by removingantibodies to shared antigenic determinants from the polyclonalantibodies before separation, only antibodies to those antigenicdeterminants not shared by the two strains will remain in the serum.This step is especially useful for identification of closely relatedstrains as it removes shared antibodies and makes the differencesbetween the strains more apparent.

The methods of the invention can be used with any type of antigen thatprovokes the production of more than one type of antibody. Thepolyclonal response to the antigen, when the antibodies are separated,represents the antigen's "fingerprint" and can be used to identify theantigen. The number of antibodies in the antigen's "fingerprint" isrelated to the number of antigenic determinants in the antigen. Aprotein, for example, would produce a "fingerprint" having fewer typesof antibodies than a larger antigen such as a virus which has a numberof proteins containing multiple antigenic determinants in its outerenvelope. Examples of protein antigens are enzymes, such asneuraminidase; blood coagulating proteins such as hemagglutinin; andviral envelope proteins, such as described for avian influenza virusherein.

The invention also provides further methods of detecting an antigen in atest sample suspected of containing the antigen. A test sample suspectedof containing the antigen is bound to a solid support. The test sampleis then contacted with polyclonal antibodies specific for the antigen,the antibodies having a detectable label bound thereon, under conditionsselected to allow binding of the antibodies to the antigen in the tissuesample. The detectable label is then detected, thereby identifying theantigen.

The methods may further comprise the step of quantitating the amount ofthe antigen present in the test sample. The methods may also furthercomprise the step of removing antibodies cross-reactive with antigensother than said antigen from the polyclonal antibodies before contactingthe test sample with the antibodies. These methods are suitable for usewith detectable labels and detection systems and methods known in theart. A preferred detectable label is biotin and a preferred method ofdetection of the detectable label comprises the steps contacting thebiotin with streptavidin-alkaline phosphatase under conditions selectedto allow the streptavidin-alkaline phosphatase to bind to the biotin;contacting the streptavidin-alkaline phosphatase with a chromogenicsubstrate; and detecting a reaction product of the chromogenicsubstrate. A preferred method of quantitating the detectable label is bydensitometric measurement. A preferred solid support is nitrocellulose.

The methods of the invention are suitable for use with antigens whichprovoke the production of more than one type of antibody. Examples ofsuitable antigens are proteins such as viral envelope proteins,infectious organisms and other molecules which provoke an immunogenicresponse. A preferred antigen is viral protein; a preferred virus isavian influenza virus. If the substance to be detected is not itselfantigenic, it can be made antigenic by conjugation to a carriermolecule, such as bovine serum albumin, using conventional techniques.

The invention further provides kits for the detection of an antigen intest samples suspected of containing the antigen. The kits compriseseparated polyclonal antibodies of the antigen bound to a solid support;a reagent comprising antibodies specific for the antigen having adetectable label bound thereon: at least one reagent comprising meansfor detecting for the detectable label; and a reference standard of theantigen comprising polyclonal antibodies of the antigen bound to a solidsupport, the antigen bound to the polyclonal antibodies and the antigendetected by the reagents of the kit. The form of the kit will depend onthe assay to be performed. For the "fingerprinting" assay, thepolyclonal antibodies would be separated and bound to a solid support.The solid support would be nitrocellulose or other suitable solidsupport. Each kit would contain one or more antigen "fingerprints". Foreach antigen to be tested, the reagent would contain antibodies for theantigen, the antibodies having a detectable label bound thereon. Theantibodies for each antigen may be supplied together in one container orin separate container. The detectable label may be any of those known inthe art for detection of antibodies which does not substantiallyinterfere with the function of the antibody to detect the virus. Thereagent for comprising means for detecting the detectable label would becoordinated with the detectable label to form a detection system for theantibodies. Some suitable detection systems may require more than onereagent for detection of the detectable label, in which case theadditional reagents would also be supplied in the kits. Examples ofsuitable detectable labels and detection systems are those describedherein. A kit for the detection of a virus, such as a strain of avianinfluenza virus, would comprise separated polyclonal antibodies specificfor that strain bound to a solid support; a reagent comprisingantibodies specific for the strain of avian influenza virus having adetectable label such as streptavidin-alkaline phosphatase conjugatebound thereon; a reagent comprising nitroblue tetrazolium (NBT); areagent comprising 5-bromo-4-chloro-3-indolyl phosphate (BCIP); and areference standard comprising polyclonal antibodies of the viral strainbound to a solid support, the virus bound to said polyclonal antibodiesand said viral strain detected by the reagents of the kit. The reagentsof the kit are designed to be used in sequence to achieve the desiredcolor.

The invention also provides other kits for detecting an antigen in atest sample suspected of containing the antigen. The kits comprise asolid support onto which the sample suspected of containing an antigenis applied, a reagent comprising antibodies specific for said antigenhaving a detectable label bound thereon; at least one reagent comprisingmeans for detecting the detectable label; and a reference standard ofthe antigen comprising the antigen bound to a solid support and detectedby the reagents in the kit.

Suitable solid supports are nitrocellulose and other solid supportsknown in the art. The reagent containing antibodies specific for theantigen may contain antibodies for one or more antigens, depending onwhether the kit is being provided to detect antigens. If the kit isdesigned to detect more than one antigen, the reagent containingantibodies having a detectable label bound thereon may be supplied inseparate containers for each antigen. The reagent comprising thesubstrate for the detectable label would be coordinated with thedetectable label to form a detection system. Some suitable detectionsystems may require more than one reagent for detection of thedetectable label, in which case the additional reagents would also besupplied in the kits. Examples of detectable labels and detectionsystems suitable for use in the reagents of the kit are those describedherein. Suitable solid supports are nitrocellulose and other solidsupports known in the art. The methods of the invention, which are to beperformed by the reagents included in the kit, are performed on thesolid support.

For both the "fingerprinting" assay and the dot blotting assay, if thekit is to be used in the field away from the laboratory, a chromogenicdetection system, such as a system employing alkaline phosphatase ispreferred. For example, separate vials containing streptavidin-alkalinephosphatase conjugate in buffer, nitroblue tetrazolium (NBT), and5-bromo-4-chloro-3-indolyl phosphate (BCIP), which are designed to beused in sequence to achieve the desired color could be supplied as partof the kit. With chromogenic detection systems such as these, theantibodies can be visualized by eye without the aid of any equipment.With chromogenic detection systems, the kit can be used in the field,and the solid support, containing the test performed using the kit ofthe invention later transported to a laboratory for quantitation of theamount of virus present in the test sample.

If it is desired to quantitate the amount of antibody present and thusthe amount of virus present when performing the methods of theinvention, a detection system employing alkaline phosphatase ispreferred. As shown herein, a system employing alkaline phosphatase willgive accurate quantitative results when used in conjunction with adensitometer.

In general, however, the detectable label may be any ligand or haptenwhich is capable of being detected, directly or indirectly and therebyindicate that binding of the antibody to the virus has occurred. Haptensare compounds having a molecular weight of approximately 6,000 or lessand which are not large enough to provoke an immunogenic response, butwhich need to be conjugated to a carrier substance in order to provokethe immunogenic response. Haptens are thus antigenic determinants andmay be adsorbed by isolated antibodies specific to them, even though thehaptens are not conjugated to the carrier. A preferred label is biotinand it may be detected as described herein. Other methods of detectingbiotin known in the art are also suitable for use in the methods of theinvention. The detectable label may also be an enzyme capable of causinga color change in a substrate solution. Suitable enzymes are horseradish peroxidase and alkaline phosphatase. The detectable label mayalso be a radioactive molecule such as³² P, ¹²⁵ I or ³ H, a fluorescentmolecule, such as fluorescein, a chemilluminescent molecule, such asluciferin or a light scattering molecule, such as colloidal gold.Suitable detection methods and systems are scintillation counting,autoradiography, fluorescence measurement, colorimetric measurement orlight emission.

Polyclonal antibodies suitable for use in the methods of the inventionmay be produced and purified by conventional antibody productionmethods. Rabbits and mice are suitable animals for production ofantibodies. Larger animals such as goats and sheep are also suitable iflarger quantities of antibodies are needed.

Examples of test samples suspected of containing an antigen which aresuitable for use in the methods and kits of the invention are bodyfluids, body tissues such as lung, spleen and kidneys, and other samplessuspected of containing antigens which are soluble or may be suspendedin a suitable fluid, such as a buffer, for use in the methods and kits.

Avian influenza virus may be obtained from chickens infected with thevirus. Alternatively, the virus may be obtained from governmentlaboratories such as the National Veterinary Services Lab, Ames, Iowa.

The solid support used in the methods of the invention may be made ofany material to which antibodies and the antigen are capable of binding,but which does not substantially interfere with the detection of theantigen. Examples of suitable supports are nitrocellulose, unmodifiednylon, cationized nylon carrying a quaternary amino charge (Zeta probe),and aminophenylthioether (APT) paper which is converted to DPT, thediazo derivative (this cannot be stained for use with enzyme detectablelabels). The solid support generally will be in the shape of a strip orsheet, but other shapes are suitable.

The methods of the invention are conveniently performed withconventional commercially-available apparatus. For quantitating theamount of detectable label present, and thus the amount of antigenpresent, the procedure and apparatus of Hazelgrove et al., Anal. Biochem150:449-456, 1985) are preferred. This procedure is based on a TV cameralinked to a computer. The dots are displayed on a light box imaged bythe TV camera, and digitized with a digitizing board (Techmar, Inc.).After digitizing, the computer will readout the position, width, heightand relative area of each dot. Optical density (OD) measurements areplotted against absolute protein concentrations.

EXAMPLES Growth and purification of influenza virus subtypes

An apathogenic strain, H5N2, (A/Mal/189/82), was provided by Dr. RobertG. Webster. Other avian apathogenic subtypes, H6N8,(A/Ty/Mn/6020-1604/81, H2N7 (A/Ty/Mn/1136/86), HlNl (A/Ty/Ks/4880/80);the index case virus for the Pennsylvania outbreak of 1983, H5N2(A/CK/PA/83); H5N2 (A/Ty/Mn/87); H7N3 (A/Ty/Or/71), as well as a numberof H5N2 isolates were sent by Dr. Jim Pearson, National VeterinaryServices Lab (NYSL), Ames, Iowa.

The viruses were grown in chicken embryos, purified by elution fromchicken erythrocytes, followed by sedimentation through a sucrosegradient, 10% through 40%, 0.15M NaCl, following the procedure of Laver,W.G., "Purification of influenza virus", in K. Habel and N.P. Salzman(eds.) Fundamental Techniques in Virology, pp.82-86, Academic Press,Inc., 1969. Lack of associated chick proteins was seen in the viruspreparations as determined by ELISA, since anti-chicken ovalbuminantibodies, sensitive at 1-5 nanograms, did not detect any ovalbuminfrom chick embryos, in the purified virus preparations, using the dotblot assay for chicken albumin.

PREPARATION OF ANTIBODIES Immunization of rabbits

Rabbits were injected intradermally with virus (hemagglutinin titers1:512-1:1024) initially in Freund's complete adjuvant, followed by virusin incomplete Freund's adjuvant at biweekly intervals.

Purification of anti-viral antiserum

Purified viruses are covalently linked to the solid matrix, Affi-Gel 10(Bio-Rad Laboratories, Richmond, CA), which consists of agarose beadscontaining the spacer N-hydroxy-succinimide. Rabbit anti-viral antiseraare absorbed to columns containing the virus-bead conjugate, the beadsare washed extensively, and the purified antibodies are eluted inglycine-HCl, pH 2.3, following the procedure of Wolf et al.,"Non-allelic inheritance of VH region group a allotypes: cell surfaceand serum studies in double and triple expressing rabbits", J.Immunology 123: 1858-1863,(1979).

Cross absorption of anti-viral antiserum

Antiviral antisera are also purified by cross-absorption for whicheither non-biotinylated or biotinylated IgG are mixed with 100 ul of asuspension of a virus (40 nanograms/100 ul), this virus being of a typedifferent than the virus which was used to make the antiserum. Themixture is incubated at 37° C. for 2 hours and overnight at 4° C., afterwhich the non-absorbed antibodies are separated after centrifugation ofvirus-antibody complex at 100,000 ×g. This procedure has been useful inderiving antibodies for private epitopes or antigenic determinants foundon a particular viral subtype, after elimination of antibodies to publicepitopes or those antigenic determinants shared by both viruses.

Biotinylation of affinity-purified antiviral antibodies orImmunoglobulin (IgG)

The antibodies or IgG (1 mg in 1 ml in 0.lM NaHCO₃) are reacted withbiotin-N-hydroxy succinimide ester, dissolved (200 ug/200ul) in dimethylsulfoxide. The mixture remains at room temperature for 4 hours and thisis dialyzed against PBS (0.15M NaCl, 0.15M potassium phosphate, pH 7.2),overnight.

Dot blot assay

The virus preparation is spotted onto a strip of nitrocellulose(Schleicher and Schuell, BA 85) in 1 ul amounts and the paper allowed todry. The blocking solution (BLOTTO, nonfat dry milk "Bovine LactoTransfer Technique Optimizer 2.5%, (2.5% w/v nonfat dry milk, 0.01%antifoam A, Sigma Chemical Co., St. Louis, Mo.) is added for 30 minutesat 37° with agitation. The nitrocellulose strips are washed 3 times withTris buffered saline (Tris, 10 mM, pH 8.0, Nacl 150 nM, Tween 20, 0.05%(TBST). The rabbit biotinylated anti-viral IgG is then added (50 ug in 2ml), incubated for 1 hr/37°, then washed with TBST, 3 times.Streptavidin-alkaline phosphatase (SAAP)(Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa.), 1 mg/ml, 1:7500, is added and thestrips are incubated for 30 minutes at room temperature after washingwith veronal acetate (0.15M, pH 9.6), 3 times. The coloring solution isadded: 5-bromo- 4 chloroindoxyl phosphate (BCIP), 5 mg/ml, 0.1 ml,nitroblue tetrazolium, 0.1%, 1 ml, MgCl₂, 2 M, 20 ul, and veronalacetate buffer, 9 ml. The nitrocellulose strips are agitated and thedots usually appear within 5 minutes. The nitrocellulose strips arewashed once in dH₂ O and dried. The strips may be stored for use asreference standards, such as might be done with a kit for detection ofavian influenza virus, or the strips ca be used for quantitation ofvirus.

Quantitation of virus

Quantitation of the amount of avian influenza virus in the sample may bedone by densitometry following the procedure of Hazelgrove et al., Anal.Biochem 150:449-456, 1985). This procedure is based on a TV cameralinked to a computer. The dots are displayed on a light box imaged bythe TV camera, and digitized with a digitizing board (Techmar, Inc.).After digitizing, the computer will readout the position, width, heightand relative area of each dot. Optical density (OD) measurements areplotted against absolute protein concentrations.

In FIG. 1, various dot blots derived from several different experimentsare represented. FIG. 1a shows the relationship of color intensity andnanogram concentration of H5N2/Mal/NY/82 when probed with homologousbiotinylated anti-H5N2 antibodies. Dots in FIG. 1 a (1) through (7)represent 10 ng, 5 ng, 2 ng, 1 ng, 0.5 ng, 0.25 and 0 ng virus protein,respectively. (In FIG. 1, the concentrations of virus are contained in 1ul of blotted suspension). FIG. 1b shows the relationship of colorintensity and nanogram concentration of H6N8/Ty/Mn/83 when probed withhomologous biotinylated anti H6N8 antibodies. Dots in FIG. 1b (1)through (7) represent 20 ng, 10 ng, 5 ng, 2 ng, 1 ng, 0.5 ng and 0 ngvirus protein, respectively. FIGS. 2a and 2b show the quantitativerepresentation of viral protein after computer-analyzed digitation. Ascan be seen, 1-2 ng of H5N2 and 1-5 ng of H6N8 could be detected; thecurves are linear.

In FIG. 1c, in order to test specificity of antibody, the purified H5N2virus was initially dot blotted at 10 ng, 5 ng, 2 ng, and 1 ngconcentrations, using the anti-H5N2 antibody FIG. 1c (1). In theadjacent panel in FIG. 1c(2), the biotinylated antibody was partiallyblocked with purified virus (8 ng virus absorbed to 450 ng antibody, andfully blocked with 16 ng virus in FIG. 1c(3).

In FIG. 1d, to test for antibody specificity, the purified H6N8 viruswas dot blotted at concentrations ranging from 10 ng, 5 ng, 2 ng, and 1ng/ul, using the homologous, biotinylated rabbit anti-H6N8 antibodypreparation as shown in FIG. 1d(1). In the adjacent panel [FIG. 1d(2)],the biotinylated antibody was partially blocked with purified virus (8ng virus absorbed to 450 ng/antibody), and fully blocked with 16 ngvirus as shown in FIG. 1d(3).

Similarly, in FIG. 1e, using the H2N7 dot blot system (FIG. 1e (1), dotscould be developed at I0 ng, 5 ng, and 1 ng/ul, and specific blocking ofthe biotinylated antibody with specific virus could be achieved panel,FIG. 1e(2)].

Fingerprinting Assay To Determine Viral Subtype Isoelectric Focusing andTransblotting Of Serum Proteins and Antibodies

Polyclonal rabbit antisera are subjected to isoelectric focusing in flatbeds of polyacrylamide of o.7 m thickness, pH range 5.5 to 8.0, 3 hours,2° C., following the method of Wolf et al., "Clonal Diversity andHomology of Latent and Nominal Group a Immunoglobulin Allotypes in theRabbit", Mol. Immunol. 21: 139-146, (1984). The focused proteins aretransblotted to nitrocellulose paper (BA85, Schleicher and Schuell)overnight, by anodal or cathodal electrotransfer (50V, 2 mA), in 25 nMTris, 192 mM glycine, pH 8.3 and 20% (v/v) methanol.

Probing the Transblotted Antibodies With Virus Followed By BiotinylatedAnti-viral Antibodies (Sandwich Technique)

The nitrocellulose paper is removed from the transblotting cassette.Strips approximately 0.5 cm wide are cut along the migration path of theisoelectrofocused proteins. The strips are laid into troughsapproximately 1.0 cm×1.0 cm×20 cm in a processing tray (Schleicher andSchuell). In succession the following steps are followed: blockingsolution, 1% bovine serum albumin (BSA), 2 ml is added and the tray isrocked for 2 hours. The troughs are then drained thoroughly. At thispoint the strips may be used immediately to test for the presence of thevirus or they may be dried and stored for future use, such as would bedone with a kit for detection of avian influenza virus.

A 1:100 dilution of virus in PBS (for example H5N2, hemagglutinin titer1:64) is added, and rocked for 1 hr/37° C. The wells are drained,biotinylated anti-H5N2 IgG is added (2 ml containing 20 ug), andincubated 1 hr/37° C. The wells are again drained andstreptavidin-alkaline phosphatase is added (10 ug/ml), and incubated atroom temperature, 30 minutes. The strips are washed with veronal buffer(3 times), the coloring solution, 5-bromo-4-chloroindoxyl phosphate(BCIP), 5 mg/ml, 0.1 ml, plus nitroblue tetrazolium (NBT), 0.1%, 1 ml,MgCl₂, 2M, 20 ul and veronal acetate buffer, 9 ml, is added, (as in theDot Blot method), with the bands appearing in 5-10 minutes. Thenitrocellulose strips are washed in distilled H₂ O and air dried. Thebands may be quantitated using densitometry as described herein,following the procedure of Haselgrove supra.

In FIG. 3 (A) is seen a blot depicting the ability of hyperimmune serafrom two different rabbits (R406 and 8401Al) to react withH5N2/Mal/NY/82 virus. After transblotting at the cathode the virus wasabsorbed to the nitrocellulose strips and probed with biotinylatedantibodies from the same two rabbits. Major bands developed, mainlybetween a pI range of 6.5 to 7.1, representing spectroptypes to virusepitopes. In lanes 4 and 5 the first eight bands, 1, 2a, 2b, 3 4, 5, 6and 7, descending from the pI of 7.1 to 6.4 have been quantitativelycomputer-digitized and the line and bar graphs are represented in FIG.4a and 4b (lane 4) and FIGS. 4c and 4d (lane 5).

In FIG. 3(b), three blots of three different subtypes of avian influenzavirus, are compared for epitope differences. The single antiserum toH5N2/Mal/Ny/82 was used to analyze each blot in FIG. 3. Afterisoelectric focusing and transblotting at the anode, the 3 differentviruses were probed with the same biotinylated anti H5N2 antibodies.

Epitope differences in bands may be seen as well as identitiesthroughout the blots. H6N8 shows band deficiencies (bands are not seenin comparison to H5N2) at pIs of 6.5, 6.3, 6.0 and 5.6. H2N7 shows banddeficiencies at pIs of 6.5, 6.3, 6.0, 5.9, 5.7 and 5.6.

It is within the scope of the invention to test for virtually anyantigen. Viral components such as hemagglutinins and neuraminidases showdifferences among subtypes and strains. Polyclonal antibodies to theseviral components would be expected to show differences between subtypesand strains based on these differences. Polyclonal antibodies toappropriately chosen viral components would then yield a "fingerprint"of the viral component for each subtype or strain. These could then beused in the fingerprint assay as described herein, alone or incombination with antibodies specific for other viral components or wholevirus.

Purification of hemagglutinin (HA)

After purification, the virus is dialyzed against 0.005M sodiumphosphate buffer, pH 7.2, then extracted at 1 mg protein/ml in the samesuffer containing Triton X-100 at 2%. The extract is separated fromundissociated viral components at 100,000 xg/60 minutes. The extract isloaded to a DEAE-agarose column and eluted with a gradient from 0-0.2MNaCl, the protein being eluted mainly at 0.06M NaCl as monitored bySDS-PAGE, following the method of Wabuke-Bunoti et al, "Stimulation ofAnti-Influenza Cytolitic T Lymphocytes by CNBr Cleavage Fragments ofViral Hemagglutinin", J. Immunol. 127: 1122-1125, (1981). Purity isconfirmed by ELISA using antisera provided by D. Senne, USDA, Ames,Iowa; these antisera may also be employed for purposes of affinitypurification of the HA, if contaminants are seen following biochemicalpurification.

Purification of neuraminidase

Purified viruses suspended in 2% NH₄ Cl, 0.01M (NH₄)₂ HPO₄ are disruptedwith ammonium deoxycholate (final concentration, 0.2%), in the absenceof Na+. The virus particles are allowed to stand for 5 hours/roomtemperature. Deoxycholate is dialyzed out. The proteins are pelleted at200,000 xg at 6 hours. The pellet is resuspended in phosphate bufferwith sonication. Nucleocapsids are separated on cellulose acetate stripsand the neuraminidase is eluted from polyacrylamide gel after separationfrom the hemagglutinin. Purity of the neuraminidase is confirmed usingantisera provided by D. Senne and anti-neuraminidase antisera may alsobe employed for purposes of affinity purification of the neuraminidase.

Quantitation of the methods of the invention would extend applicationsfor the methods. For example, shared epitopes may be quantitated notonly among viral subtypes, but also among strains within a viralsubtype. Further the methods are highly sensitive. The level ofsensitivity of the dot blot is at 1-5 ng antigen and in the"fingerprinting" assay, fewer than 10 ng of antigen protein may bedetectable.

What is claimed is:
 1. A method for detecting an antigen in a testsample suspected of containing said antigen and simultaneouslydetermining a fingerprint of antibodies specific for said antigen,comprising the steps:(a) providing polyclonal antibodies specific forsaid antigen; (b) separating said polyclonal antibodies specific forsaid antigen from each other according to the electrical charge ofindividual antibodies comprising said polyclonal antibodies; (c) bindingsaid antibodies thus separated to a solid support such that saidantibodies separated in step (b) maintain the same relative positionwith respect to each other on said solid support to thereby form afingerprint of antibodies specific for said antigen; (d) contacting saidantibodies bound in step (c) with a test sample suspected of containingsaid antigen under conditions selected to allow binding of said antigento said antibodies bound in step (c); (e) contacting antigen bound instep (d) with detectably labeled antibodies specific for said antigen,under conditions selected to allow binding of said detectably labeledantibodies to said antigen; (f) detecting said detectably labeledantibody to thereby indicate the presence of said antigen in said testsample and reveal said fingerprint of antibodies specific for saidantigen when said antigen is present in said test sample.
 2. The methodof claim 1 further comprising the step of quantitating the amount ofsaid labeled antibody bound to said antigen and relating said amount tothe amount of said antigen present in the test sample.
 3. The method ofclaim 2 wherein the step of quantitating is be densitometricmeasurement.
 4. The method of claim 1 further comprising the step ofremoving antibodies cross-reactive with antigens other than said antigenfrom said polyclonal antibodies before step (b).
 5. The method of claim1 wherein said detectably labeled antibody is labeled with biotin. 6.The method of claim 5 wherein said detectably labeled antibody isdetected by(a) contacting said biotin with streptavidin-alkalinephosphate under conditions selected to allow the streptavidin-alkalinephosphatase to bind to said biotin; (b) contacting saidstreptavidin-alkaline phosphatase with a chromogenic substrate suitablefor alkaline phosphatase; and (c) detecting a reaction product of saidchromogenic substrate.
 7. The method of claim 1 wherein said antigen isa viral protein.
 8. The method of claim 7 wherein said viral protein isan avian influenza viral protein.
 9. The method of claim 1 wherein saidsolid support is nitrocellulose.
 10. The method of claim 1 wherein thestep of separating said polyclonal antibodies is performed byisoelectric focusing.
 11. A kit for identification of an antigen in atest sample suspected of containing said antigen, comprising:(a) a solidsupport having bound thereon polyclonal antibodies specific for saidantigen, said antibodies having been separated from each other accordingto the electrical charge of the individual antibodies comprising thepolyclonal antibodies and bound to said solid support such that theseparated antibodies maintain the same relative position with respect tothe each other on said solid support; (b) a reagent comprisingantibodies specific for said antigen labeled with a detectable label;(c) at least one reagent comprising means for detecting said detectablelabel in (b); and (d) reference standard of said antigen.
 12. The kit ofclaim 11 wherein said solid support is nitrocellulose.
 13. The kit ofclaim 11 wherein the separation of said polyclonal antibodies isachieved by isoelectric focusing.
 14. The kit of claim 11 wherein saidantigen is a viral protein.
 15. The kit of claim 23 wherein said viralprotein is an avian influenza viral protein.
 16. A method for detectingan antigen in a test sample suspected of containing said antigen andsimultaneously determining a fingerprint of antibodies specific for saidantigen, comprising the steps:(a) providing a solid support having boundthereon polyclonal antibodies specific for said antigen, said antibodieshaving been separated from each other according to the electrical chargeof the individual antibodies making up the polyclonal antibodies andbound to said solid support such that the said antibodies thus separatedmaintain the same relative position with respect to each other on saidsolid support to thereby form a fingerprint of antibodies specific forsaid antigen; (b) contacting said solid support with a test samplesuspected of containing said antigen under conditions selected to allowbinding of said antigen to said antibodies bound on said solid support;(c) contacting said antigen thus bound on said solid support in step (b)with detectably labeled antibody specific for said antigen underconditions selected to allow binding of said antibody to said antigen;(d) detecting said detectably labeled antibody thus bound in step (c) tothereby indicate the presence of said antigen in said test sample andreveal said fingerprint of antibodies specific for said antigen whensaid antigen is present in said test sample.
 17. The method of claim 16further comprising the step of quantitating the amount of saiddetectably labeled antibody bound to said antigen and relating saidamount to the amount of said antigen present in the test sample.
 18. Themethod of claim 17 wherein the step of quantitating is by densitometricmeasurement.
 19. The method of claim 29 wherein said detectably labeledantibody is labeled with biotin.
 20. The method of claim 32 wherein saiddetectably labeled antibody is detected by(a) contacting said biotinwith streptavidin-alkaline phosphatase under condition selected to allowthe streptavidin-alkaline phosphatase to bind to said biotin; (b)contacting said streptavidin-alkaline phosphatase with a chromogenicsubstrate suitable for alkaline phosphatase; and (c) detecting areaction product of said chromogenic substrate.
 21. The method of claim16 wherein said antigen is a viral protein.
 22. The method of claim 21wherein said viral protein is an avian influenza viral protein.
 23. Themethod of claim 16 wherein said solid support is nitrocellulose.
 24. Themethod of claim 1 further comprising the step ofcomparing saidfingerprint of antibodies of step (f) with a fingerprint of antibodiesspecific for a reference standard of said antigen.
 25. The method ofclaim 16 further comprising the step ofcomparing said fingerprint ofantibodies of step (d) with a fingerprint of antibodies specific for areference standard of said antigen.